Manual innuprep Virus DNA/RNA Kit - IPC96

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1 Manual

2 Order No.: 845-IP IP reactions 480 reactions Publication No.: HB_IP-5096_e_ This documentation describes the state at the time of publishing. It needs not necessarily agree with future versions. Subject to change! Expression and further use permitted with indication of source. Copyright 2015, Analytik Jena AG, AJ Innuscreen GmbH Manufacturer: AJ Innuscreen GmbH Robert-Rössle-Straße Berlin Made in Germany! Distribution/Publisher: Analytik Jena AG Konrad-Zuse-Straße Jena/Germany Phone +49 (0) / Fax +49 (0) / lifescience@analytik-jena.com

3 Contents Contents 1 Introduction Intended use Notes on the use of this manual Safety precautions Storage conditions Function testing and technical assistance Product use and warranty Kit components Recommended steps before starting Components not included in the kit Product specifications General notes and safety recommendations on handling RNA Principle of operation Carrier Mix Storage conditions and handling Preparation of Lysis Solution RL / Carrier Mix Lysis protocols for isolation of viral DNA and RNA from different starting materials Protocol 1: Isolation of viral DNA and RNA from cell-free body (serum, plasma, cerebrospinal fluid, liquor) Protocol 2: Isolation of viral DNA and RNA from cell culture supernatants Preliminary steps of the InnuPure C Automatic processing of the InnuPure C Related Products innuprep Virus DNA/RNA Kit-IPC96 Issue 04 /

4 Contents 2 Issue 04 / 2015 innuprep Virus DNA/RNA Kit-IPC96

5 Introduction 1 Introduction 1.1 Intended use The innuprep Virus DNA/RNA Kit - IPC96 has been designed for the fully automated isolation of both viral DNA and RNA from serum, plasma and other cell free clinical samples, from cell culture supernatants, and other relevant liquid starting materials. The extraction procedure is based on a new patented chemistry. For the liquid samples all steps of the extraction process are fully automated and run completely on the InnuPure C96. The samples are transferred into the Sample Reagent Plates of the kit, which is already prefilled with all extraction reagents needed for the extraction process. The following extraction process runs automatically on the InnuPure C96. The extraction process is based on binding of the DNA and/or RNA on surface modified magnetic particles. After washing steps the nucleic acid is eluted from the magnetic particle with RNasefree water and is now ready to use for downstream applications. The extraction chemistry in combination with the InnuPure C96 protocol are optimized to get maximum yield and quality. For controlling the extraction process the kit contains a Carrier Mix with a Carrier RNA and internal control DNA (IC DNA) and RNA (IC RNA). The IC DNA and IC RNA can be detected by real time PCR with a corresponding real time PCR detection kit. Consult instruction for use This package insert must be read carefully prior to use. Package insert instructions must be followed accordingly. Reliability of results cannot be guaranteed if there are any deviations from the instructions in this package insert. innuprep Virus DNA/RNA Kit-IPC96 Issue 04 /

6 Introduction 1.2 Notes on the use of this manual For easy reference and orientation, the manual uses the following warning and information symbols as well as the shown methodology: REF Catalogue number. N Content Contains sufficient reagents for <N> reactions. Storage conditions Store at room temperature or shown conditions respectively. Consult instructions for use This information must be observed to avoid improper use of the kit and the kit components. Used by Expiry date. Lot number The number of the kit charge. Manufactured by Contact information of manufacturer. For single use only Do not use components for a second time. Note / Attention Observe the notes marked in this way to ensure correct function of the device and to avoid operating errors for obtaining correct results. The following systematic approach is introduced in the manual: The chapters and figures are numbered consecutively. A cross reference is indicated with an arrow (e.g. "Notes on the use of this manual" p. 4). Working steps are numbered. 4 Issue 04 / 2015 innuprep Virus DNA/RNA Kit-IPC96

7 Safety precautions 2 Safety precautions Note Read through this chapter carefully prior to guarantee your own safety and a trouble-free operation. Follow all the safety instructions explained in the manual, as well as all messages and information, which are shown. All due care and attention should be exercised in handling the materials and reagents contained in the kit. Always wear gloves while handling these reagents and avoid any skin contact! In case of contact, flush eyes or skin with a large amount of water immediately. For single use only! This kit is made for single use only! Attention! Don t eat or drink components of the kit! The kit shall only be handled by educated personal in a laboratory environment! If the buffer bottles are damaged or leaking, wear gloves and protective goggles when discarding the bottles in order to avoid any injuries. Analytik Jena AG has not tested the liquid waste generated during using the kit for potential residual infectious components. This case is highly unlikely, but cannot be excluded completely. Therefore, all liquid waste must be considered as potentially infectious and must be handled and discarded according to local safety regulation. Please observe the federal, state and local safety and environmental regulations. Observe the usual precautions for applications using extracted nucleic acids. All materials and reagents used for DNA or RNA isolation should be free of DNases or RNases. Attention! Do not add bleach or acidic components to the waste after sample preparation! Note Emergency medical information in English and German can be obtained 24 hours a day from: Poison Information Center, Freiburg / Germany Phone: +49 (0) For more information, please ask for the material safety data sheet (MSDS): innuprep Virus DNA/RNA Kit-IPC96 Issue 04 /

8 Storage conditions 3 Storage conditions The innuprep Virus DNA/RNA Kit IPC96 should be stored dry, at room temperature (14 25 C) and is stable for at least 6 months under these conditions. Before every use make sure that all components have room temperature. If there are any precipitates within the additional provided solutions solve these precipitates by careful warming. For further information see table kit components ( "Kit components", p. 7). 4 Function testing and technical assistance The Analytik Jena AG guarantees the correct function of the kit for applications as described in the manual. The components of each innuprep Virus DNA/RNA Kit IPC96 were tested by recovery of IC DNA and IC RNA spiked in serum in serial dilutions and subsequent real-time PCR. We reserve the right to change or modify our products to enhance their performance and design. If you have any questions or problems regarding any aspects of the innuprep Virus DNA/RNA Kit IPC96 or other Analytik Jena AG products, please do not hesitate to contact us. For technical support or further information in Germany please dial / For other countries please contact your local distributor. 5 Product use and warranty The user is responsible to validate the performance of the Analytik Jena AG kits for any particular use, since the performance characteristics of our kits have not been validated for any specific application. Analytik Jena AG kits may be used in clinical diagnostic laboratory systems after the laboratory has validated the complete diagnostic system as required by CLIA 88 regulations in the U.S. or equivalents in other countries. All products sold by the Analytik Jena AG are subjected to extensive quality control procedures and are warranted to perform as described when used correctly. Any problems should be reported immediately. All due care and attention should be exercised in handling the materials and reagents contained in the kit. Always wear gloves while handling these reagents and avoid any skin contact! In case of contact, flush eyes or skin with a large amount of water immediately. Use the kit once only! For skilled personal only! Don t eat or drink components of the kit! Note For research use only! 6 Issue 04 / 2015 innuprep Virus DNA/RNA Kit-IPC96

9 Kit components 6 Kit components Important Store lyophilized Proteinase K at 4 C! Divide dissolved Proteinase K into aliquots and storage at 20 C is recommended. Repeated freezing and thawing will reduce the activity dramatically! Store lyophilized Carrier Mix at 20 C. Storage conditions All other components are stored at room temperature IP IP Lysis Solution RL 30 ml 160 ml Proteinase K For 2 x 1.5 ml working solution For 7 x 1.5 ml working solution Carrier Mix 1x lyophilized powder 5x lyophilized powder RNase-free water 1x 2 ml 5x 2 ml Plate Sample Plate (empty) Plate MAG Suspension E Plate Binding Solution VL Plate Washing Solution HS Plate Transfer Plate (empty) Plate RNase-free Water Plate Washing Solution LS Plate RNase-free Water Plate Elution (RNase-free Water) Plate 10 Transfer Plate (empty) 1 5 innuprep Virus DNA/RNA Kit-IPC96 Issue 04 /

10 Kit components 845-IP Plate Elution Plate (empty) Filter Tips 1x 96 5x 96 Manual 1 1 Initial steps Proteinase K: Dissolve Proteinase K by addition of 1.5 ml of ddh 2 O, mix thoroughly and store as described above! Carrier Mix: Add 1.25 ml RNase-free Water to the tube Carrier Mix, mix thoroughly by pipetting up and down! 845-IP Proteinase K: Dissolve Proteinase K by addition of 1.5 ml of ddh 2 O, mix thoroughly and store as described above! Carrier Mix: Add 1.25 ml RNase-free Water to each tube Carrier Mix, mix thoroughly by pipetting up and down! 8 Issue 04 / 2015 innuprep Virus DNA/RNA Kit-IPC96

11 Recommended steps before starting 7 Recommended steps before starting! Important Invert the Reagent Plates for 3 4 times and thump it onto a table to collect the pre-filled solutions at the bottom of the strips. Ensure that the Proteinase K and Carrier Mix have been prepared according to the instruction ( "Kit components", p. 7). Ensure that the Carrier Mix and Lysis Solution RL / Carrier Mix have been prepared according to the instruction ( "Carrier Mix" p. 13). Centrifugation steps should be carried out at room temperature. Avoid freezing and thawing of starting material. 8 Components not included in the kit ddh 2 O 1.5 ml reaction tubes 9 Product specifications 1. Starting material: cell-free body samples (serum, plasma, cerebrospinal fluid, liquor; max. volume 200 µl) cell culture supernatants (max. volume 200 µl) 2. Time for isolation: Lysis: automatic on device InnuPure C96 protocol standard: approx: 84 min innuprep Virus DNA/RNA Kit-IPC96 Issue 04 /

12 General notes and safety recommendations on handling RNA 10 General notes and safety recommendations on handling RNA RNA is far less stable than DNA. It is very sensitive to degradation by endogenous RNases in the biological material and exogenous RNases which are permanently present everywhere in the lab. To achieve satisfactory qualitative and quantitative results in RNA preparations, contaminations with exogenous RNases have to be reduced to a minimum in accordance with the following recommendations: Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contaminations from surface of the skin or from dusty laboratory equipment. Change gloves frequently and keep tubes closed. Keep isolated RNA on ice. Reduce preparation time as much as possible. Use only sterile, disposable polypropylene tubes throughout the procedure (these tubes are generally RNase-free.) Non-disposable plastic ware should be treated before use to ensure that it is RNase-free. Plastic ware should be thoroughly rinsed with 0.1 M NaOH, 1 mm EDTA followed by RNase-free water. You can also take chloroformresistant plastic ware rinsed with chloroform to inactivate RNases. All glassware should be treated before use to ensure that it is RNase-free. Glassware should be cleaned with detergent, thoroughly rinsed and oven baked at 240 C for four or more hours before use. Autoclaving alone will not inactivate many RNases completely. Oven baking inactivates RNases and ensures that no other nucleic acids (such as plasmid DNA) are present on the surface of the glassware. You can also clean glassware with 0.1 % DEPC (diethyl pyrocarbonate). The glassware has to be immersed in 0.1 % DEPC solution for 12 hours at 37 C and then it has to be autoclaved or heated to 100 C for 15 min to remove residual DEPC. Electrophoresis tanks should be cleaned with detergent solution (e.g. 0.5 % SDS), thoroughly rinsed with RNase-free water, rinsed with ethanol and finally allowed to dry. All buffers have to be prepared with DEPC-treated RNase-free ddh 2 O. Avoid handling bacterial cultures, cell cultures or other biological sources of RNases in the same lab where the RNA purification will be performed. Do not use equipment, glassware and plastic ware employed for other applications which might introduce RNase contaminations in the RNA isolation. 10 Issue 04 / 2015 innuprep Virus DNA/RNA Kit-IPC96

13 Principle of operation 11 Principle of operation Depending on the type and features of selected starting material, a given lysis process may have to be performed inside the system (internal process) or outside the system (external process) in manual mode. Refer to the user manual of a given extraction kit for a detailed description of lysis working steps/instructions for the corresponding type of starting material. Figure Step Explanation 1. Lysis (internal) 1. Lysis (external) For internal lysis, the necessary reagents are at first added to the samples in a sample plate and the sample plate is placed onto the left carriage position. This is followed by motion to transfer the plate into its working position below the pipetting head. In the majority of cases, the heating plate is then pressed against the well bottoms in order to process thermal pulpings. Continuous mixing throughout this working step will help the lysis process. For external lysis, a sample plate is equally placed onto the left carriage position. The automatic protocol routine begins with the workstep of binding. 2. Binding The procedure of binding nucleic acids to the magnetic particles begins with the addition of binding buffer. Once the binding buffer has been transferred, a homogeneous mixture of the various involved components is achieved by way of pipetting-on and pipetting-off steps performed in quick succession. 3. Collecting To collect the magnetic particles at the plate s well bottom, the heating magnet module below the plate moves into place at the plate. It moves exactly as far as necessary for the magnets to make direct contact with the well bottom through the heating plate. Any excessive binding buffer can thus be pipetted off and discarded. 4. Washing Any residual amount of magnetic particles at the well bottom can now be solved in a washing buffer. Repeated/multiple pipetting-on and pipetting-off cycles warrant proper mixing. Magnetic particles are subsequently collected again at the well bottom and any excessive amount is transferred back into the reagent plate that contains pre-filled washing buffer. innuprep Virus DNA/RNA Kit-IPC96 Issue 04 /

14 Principle of operation This procedure is several times repeated using different types of washing buffer. The number of required washing cycles, the required volume and the type of washing buffer are conditional on the type of selected starting material. 5. Transfer pipetting Depending on the particular protocol, the complete content of a plate is transferred into a new plate during and/ or on completion of a washing routine. This distinctly improves the quality of nucleic acids already washed. Any residual amount of lysis debris at the well walls will thus stay back in the latest washed plate and only those magnetic particles which have already been washed and are bound to nucleic acids will be subject to further transportation. 6. Tip washing In order to avoid any carry-over effects of residue potentially accumulating in the tips, the tips are subjected to rinsing in between certain washing steps. 7. Ethanol removal To remove residue of ethanol inside the wells, a drying workstep follows. This workstep consists in the heating plate of the heating magnet module being turned on and pressed against the bottom of the plate. 8. Elution Elution buffer is supplied. This is followed by heating of the mixture and homogenization by performing continuous pipetting-on and pipetting-off cycles. In the process nucleic acids get detached from the magnetic particles. As part of an elution workstep magnetic particles are again collected at the well bottom and the mixture is subsequently transferred to a new plate where the remaining magnetic particles are once more collected at the well bottom. 9. Eluate transfer Once all magnetic particle residues have gathered at the well bottom, the eluate is transferred into the elution plate. The software screen reports completion of the protocol sequence and the elution plate which is located in the left carriage position can now be retrieved from the system (after opening of the left door). Note Filling levels and filling colors are only shown for illustration and do not match the actual filling levels and colors of the kit reagents. 12 Issue 04 / 2015 innuprep Virus DNA/RNA Kit-IPC96

15 Carrier Mix 12 Carrier Mix 12.1 Storage conditions and handling The Carrier Mix contains a carrier RNA and an internal control DNA and RNA (IC DNA and IC RNA). Add dissolved Carrier Mix to Lysis Solution RL immediately. Unused Carrier Mix should kept frozen at 20 C. Do not freeze and thaw the Carrier Mix more than 3 times. Mixture of Lysis Solution RL and Carrier Mix is stable for 7 days at 4 C. Internal control DNA or RNA can be detected by real time PCR using the corresponding assays, as shown below: Name Amount Order-no. Detection of IC DNA innudetect Internal Control DNA Assay 100 rxn 845-ID Detection of IC RNA innudetect Internal Control RNA Assay 100 rxn 845-ID Detection of IC DNA and IC RNA innudetect Internal Control DNA/RNA Assay 100 rxn 845-ID Preparation of Lysis Solution RL / Carrier Mix 1. Add 1.25 ml RNase-free Water to each tube Carrier Mix. 2. Mix thoroughly by pipetting up and down! 3. After the preparation of Carrier Mix stock solution prepare the mixture of Lysis Solution RL / Carrier Mix as described in the following table: Reagent 48 samples 96 samples 480 samples n x samples Lysis Solution RL 12 ml 24 ml 120 ml 240 µl x sample Carrier Mix 600 µl 1.2 ml 6.0 ml 12 µl x sample Note: If customized Extraction Controls should be used, please add these components to the Lysis Solution RL / Carrier Mix! innuprep Virus DNA/RNA Kit-IPC96 Issue 04 /

16 Lysis protocols for isolation of viral DNA and RNA from different starting materials 13 Lysis protocols for isolation of viral DNA and RNA from different starting materials Note The lysis of the starting material is done automatically and is included in the InnuPure C96 extraction protocol. Prepare Lysis Solution RL / Carrier Mix as described on page 13 before starting! 13.1 Protocol 1: Isolation of viral DNA and RNA from cell-free body (serum, plasma, cerebrospinal fluid, liquor) 1. Transfer 200 µl Lysis Solution RL / Carrier Mix into the wells of Plate 1 - Sample Plate (empty). 2. Add 200 µl of the sample to each well containing Lysis Solution RL / Carrier Mix. 3. Add 20 µl Proteinase K to each well containing Lysis Solution RL / Carrier Mix and the sample. Note The sample will be processed using the InnuPure C96. Please follow the instruction of the manual from point 15! 13.2 Protocol 2: Isolation of viral DNA and RNA from cell culture supernatants 1. Transfer 200 µl Lysis Solution RL / Carrier Mix into the wells of Plate 1 - Sample Plate (empty). 2. Add 200 µl of the cell culture supernatant (cell culture medium) to each well containing Lysis Solution RL / Carrier Mix. 3. Add 20 µl Proteinase K to each well containing Lysis Solution RL / Carrier Mix and the sample. Note The sample will be processed using the InnuPure C96. Please follow the instruction of the manual from point 15! 14 Issue 04 / 2015 innuprep Virus DNA/RNA Kit-IPC96

17 Preliminary steps of the InnuPure C96 14 Preliminary steps of the InnuPure C96! Note Be sure that the pre-filled solutions are at the bottom of the 96 Deep Well Plates. Important! Use the piercing tool to open the cavities of the 96 Deep Well Plates! Open the left door of the InnuPure C96 and load the Plate 1 - Sample Plate with the samples and Proteinase K on the left position of sample tray. Open the door of Stacker A (right Stacker) of the InnuPure C96 and load the opened pre-filled 96 Deep Well Plates (Plate 2-11) as shown below: Stacker A top Plate 11 - Elution Plate (empty) Plate 10 - Transfer Plate (empty) Plate 9 - Elution (RNase-free Water) Plate 8 - RNase-free Water Plate 7 - Washing Solution LS Plate 6 - RNase-free Water Plate 5 - Transfer Plate (empty) Plate 4 - Washing Solution HS Plate 3 - Binding Solution VL bottom Plate 2 - MAG Suspension E! Important Note - Inserting 96 Deep Well Plates Always make sure that A1 faces toward the left back-end corner as you insert a Deep Well Plate! innuprep Virus DNA/RNA Kit-IPC96 Issue 04 /

18 Automatic processing of the InnuPure C96 15 Automatic processing of the InnuPure C96 1. Check for correct connection of power cord and USB cable. Turn InnuPure C96 into stand-by mode by transferring ON/OFF switch into position I. To switch the device on, keep the touch sensor depressed for 1.5 sec (approximately) the LED will change from red to green light. 2. Turn PC on and trigger the software Application Studio. 3. Start the extraction protocol: Use the radio button in the selection window (see page 44 in the device manual). Virus_C96_03.bms Select the desired extraction protocol Virus_C96_03 and press [START] Follow the instructions of the extraction protocol. Enter the recommended eluate volume of 100 µl and press [OK]. Note: It is possible to adjust the volume values between 50 µl up to 500 µl. Find more detail information how to start an extraction protocol in InnuPure C96 Handbook 7.2 Software functions 44! 16 Issue 04 / 2015 innuprep Virus DNA/RNA Kit-IPC96

19 Automatic processing of the InnuPure C96 Choose log-file and sample-id, if is needed and press [OK] or [CANCEL]. Note: It is now possible to enter sample-id s and creation of a log-file. Find more detail information how to start an extraction protocol in InnuPure C96 Handbook 7.2 Software functions 44! 4. After completion of the protocol open the door of sample position. Note: The chosen protocol is performed by device and after the protocol is finished the message 'Purification process completed' is shown in the screen of the computer! 5. Remove the Elution Plate from the left carriage position of the InnuPure C96 with the final eluates containing the extracted DNA/RNA; close the Plate with a foil and store the DNA/RNA under proper conditions. 6. After finishing the extraction protocol, remove the used Deep Well Plates from Stacker B (left Stacker) and discard the Tip tray with used Filter Tips. Note After finishing the extraction protocol, the Elution Plate contains the extracted DNA and RNA. Store the DNA and RNA under adequate conditions. We recommend to store the extracted DNA and RNA at 20 C. For long time storage placing at -80 C is recommended! innuprep Virus DNA/RNA Kit-IPC96 Issue 04 /

20 Related Products 16 Related Products Name Amount Order No. Detection of IC DNA innudetect Internal Control DNA Assay 100 rxn 845-ID Detection of IC RNA innudetect Internal Control RNA Assay 100 rxn 845-ID Detection of IC DNA and IC RNA innudetect Internal Control DNA/RNA Assay 100 rxn 845-ID Nucleic acid purification innuprep Proteinase K 6 mg 845-CH mg 845-CH Products for Reverse Transcription/qPCR innuscript Reverse Transcriptase [25 U/µl] 50 rxn 845-RT (1.250 U) 200 rxn 845-RT (5.000 U) innuscript One Step RT-PCR SyGreen Kit 100 rxn 845-RT rxn 845-RT innuscript One Step RT-PCR Probe Kit 100 rxn 845-RT rxn 845-RT Products for PCR & Gel Electrophoresis innutaq DNA Polymerase (5 U/µl) 500 U 845-EZ innutaq RED DNA Polymerase (1 U/µl) 500 U 845-EZ innutaq Hot-A DNA Polymerase (5 U/µl) 500 U 845-EZ innutaq UltraPure DNA Polymerase (5 U/µl) 500 U 845-EZ x innucleotide Mix (12,5 mm) 2x 0.5 ml 845-AS innucleotide Set (100 mm) 4x 0.25 ml 845-AS mm MgCl 2 - Solution 3x 1.5 ml 845-AS mm MgCl 2 - Solution 3x 1.5 ml 845-AS PCR-grade H 2 O 2.0 ml 845-AS x 2.0 ml 845-AS innumix rapidpcr MasterMix 100 rxn 845-AS rxn 845-AS innumix Standard PCR MasterMix 100 rxn 845-AS rxn 845-AS innumix Green PCR MasterMix 100 rxn 845-AS rxn 845-AS Issue 04 / 2015 innuprep Virus DNA/RNA Kit-IPC96

21 Related Products Name Amount Order No. innustar 100 bp DNA Ladder Express 500 µl 845-ST x 500 µl 845-ST innustar 1 kb DNA Ladder Express 500 µl 845-ST x 500 µl 845-ST x Loading Dye Bromophenol Blue 3x 1.0 ml 845-ST x 1.0 ml 845-ST x Loading Dye Orange G 3x 1.0 ml 845-ST x 1.0 ml 845-ST Products for qpcr innumix qpcr MasterMix Probe 100 rxn 845-AS rxn 845-AS innumix qpcr MasterMix SyGreen 100 rxn 845-AS rxn 845-AS innuprep Virus DNA/RNA Kit-IPC96 Issue 04 /

22 engl. 04/15 Analytik Jena AG, Jena

23 Analytik Jena AG Life Science Konrad-Zuse-Strasse Jena / Germany Phone +49 (0) Fax +49 (0) lifescience@analytik-jena.com

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